Proteomics

A few studies have been published using proteomic approaches that involve chemical labeling of S-nitrosated proteins, affinity isolation, one or two-dimensional gel electrophore-sis, and mass spectrometry. These studies have consistently shown that a limited number of S-nitrosoproteins exist in cells treated with NO-donors, suggesting a selectivity of S-nitrosation [25,103-105]. In these reports, peroxiredoxin [25,105], actin [25,103,105], and GAPDH [25,103,105] were identified as S-nitrosoproteins. The function of peroxiredoxin relies on an active cysteine that is critical for the decomposition of peroxide [106].

Both GAPDH and actin are known to contain reactive cysteinyl residues [107]. Under physiological conditions, S-nitrosation of one or a few cysteinyl residues changes the function of OxyR [108], L-type calcium channels [109], NSF ATPase [110], and caspase [111]. These findings suggest that there is specificity to S-nitrosation of the cysteinyl functionality that depends on its pK. Typically ~8.4 in aqueous environments, the pK of cysteinyl sulfhydryl functionality can become anomalously low within the constraints of the tertiary structure of a protein and proximity to charged side-chains, especially histidine and aspartate, leading to thiolate anion formation. For example, the pK of serum albumin's single cysteinyl group (cys34) is ~4.5 [112], rendering it susceptible to electrophilic attack by the partially positively charged nitric oxide moiety of a low-molecular-weight S-nitrosothiol such as S-nitrosoglutathione [79,80,113]. These S-nitrosation-specific thiols may also change by protein allostery [114,115], controlled by binding of metal ions or other cofactors.

Rhee and colleagues studied the S-nitroso-proteome in Mycobacterium tuberculosis [116], which is a target for antimicrobial endogenous reactive nitrogen species. Twenty-nine S-nitroso proteins were identified from Mycobacterium tuberculosis that infected bone marrow-derived primary mouse macrophages, and all are enzymes important for invasion and replication of the bacterium.